This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topics, 06-AG-108 Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues, and 06-DK-105 Enabling technologies for cell biology and macromolecular analyses. The long-term health of all metazoan cells is inextricably linked to protein quality control. This is achieved by proteostasis, a complex network of molecular interactions that determines the health of the proteome. Proteostasis balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance with the challenges imposed by environmental or physiological stress that results in a flux of misfolded and damaged proteins. An imbalance in homeostasis, if left unattended can result in severe molecular damage to the cell, dysregulation of key tissues leading to pathology, and susceptibility to diseases of aging. Adaptation and survival requires an ability to sense damaged proteins and to coordinate induction of protective stress response pathways, chaperone, and clearance networks. Despite the abundance and apparent capacity of chaperones and other components of the proteostasis network to restore folding equilibrium, the cell is poorly adapted for chronic proteotoxic stress as occurs when certain aggregation-prone proteins are expressed in metabolic disease, cancer, and neurodegenerative disease. This decline in repair activities that challenges the integrity of the proteome is influenced strongly by genes that control aging thus linking stress biology, metabolism, and protein homeostasis with the health and lifespan of the organism. This proposal brings together the complementary strengths of two groups, the Dillin laboratory at the Salk Institute and the Morimoto laboratory at Northwestern University to develop and test a new set of molecular tools that will report on the health of the proteome. These "proteostasis sensors" are designed to provide real-time assessment of the capabilities of protein folding quality control in each compartment of the cell and to assess the consequences of protein damage, cell stress, aging, and diseases of protein conformation. These tools will be initially developed for use in C. elegans to obtain a rapid test of hypothesis and the ability to assess functional capacities using genetic approaches and then extended to mammalian tissue culture cells and eventually in transgenic mice. The impact of these studies is very broad and extends across all areas of biology and medicine, for protein quality control is fundamental to the health and protection of the proteome in all cells and tissues of eukaryotes. The tools that we develop will be made available to all other researchers upon request upon publication. PUBLIC HEALTH RELEVANCE: The expression of damaged proteins is associated with hundreds of human diseases associated with aging. How protein misfolding in one compartment of the cell affects another and variability among tissues represents key questions that have not been resolved. This proposal is to develop a molecular toolbox of folding sensors that provide real-time living cell imaging to quantify the health of the proteome in the face of acute stress, chronic expression of damaged proteins, and aging.